Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential

1 Division of Nephrology and Hypertension, Department of Medicine, University of Texas Health Science Center, and 2 Department of Medicine, Baylor College of Medicine, Houston, Texas 77030 Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N G -monomethyl- L -arginine ( L -NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO 3 /NO 2 production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L -NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S -nitroso- N -acetyl- D , L -penicillamine (SNAP) at a concentration (0.1 µM) that restored cGMP production to the basal value. In the presence of L -NMMA, insulin no longer affected cGMP production but when insulin was added to L -NMMA plus SNAP, cGMP production was increased by 69% ( P